Process for the preparation of estrane compound by fermentation

ABSTRACT

Estrane compounds that are known to be useful as sex hormones or as intermediates thereof are prepared by subjecting 19hydroxyandrostane starting compounds to the action of Corynebacterium equi or to the action of the enzyme produced by said micro-organism under aerobic conditions in the presence of a surface active agent of the fatty acid ester series such as Tween 80, said starting compound being incorporated into a culture medium at a concentration of 2 to 3 w/v percent.

United States Patent Shirasalra et M.

[54] PROCESS FOR THE PREPARATION OF ESTRANE COMPOUND BY FERMENTATION [72] Inventors: Makoto Shirasaka; Katsumi Tanabe; At-

sushi Naito; Masako Teki, all of Tokyo,

Japan [73] Assignee: Sankyo Company Limited, Tokyo, Japan [22] Filed: Aug. 12, 1968 [21] Appl. No.: 751,735

[52] U.S. Cl ..l95/51F, 195/117 [51] Int. Cl ..C07c 167/14 [58] Field of Search ..195/5l G, 51 F, 117

[56] References Cited UNlTED STATES PATENTS 3,162,655 12/1964 Bagli ..195/51 F 51 Feb.1,T972

3,189,528 6/1965 Smith et al ..195/51 F 3,344,038 9/1967 Greenspan et all .195/51 F 3,379,621 4/1968 Campillo ..195/51 (1 3,395,078 7/1968 Vezina et al. 195/51 G 3,451,892 6/1969 Herzog et al... ....195/51 G 3,507,749 4/1970 Sih ..195/51 0 Primary Examiner-Alvin E. Tanenholtz Attorney-McClew and Toren [57] ABSTRACT 3 Claims, N0 Drawings PROCESS FOR THE PREPARATION OF ESTRANE COMPOUND BY FERMENTATION This invention relates to a novel method for the production of an estrane compound by fermentation. 5 More particularly it relates to a novel process for preparing an estrane compound having the formula wherein R is hydroxy group and R is a lower alkynyl group or R, and R when taken with the carbon atom to which they are attached, represent keto group which comprises subjecting a l9-hydroxyandrostane compound having the formula wherein R has the same meaning as hereinabove and R' is hydrogen atom or a lower alkynyl group or R and R;., when taken together with the carbon atom to which they are attached, represent keto group, to the action of a micro-organism selected from those micro-organisms of the genera C orynebacterium and Arthrobacter or to the action of the enzymes produced by said micro-organism under aerobic condilion.

The above-mentioned estrane compound (I) is a steroidal compound which is well known in the art to be useful as ovarian hormones.

The chemical synthesis of estrone was heretofore rewarded with success [See, Journal of the Pharmaceutical Society of Japan, 80, l67l, 1960)].

Moreover, the microbiological process for the preparation of estrone is disclosed in the Journal of the American Chemical Society, 83, 4,627, l96l According to the disclosure of the latter literature, estrone is produced by subjecting l9- hydroxyandrost-4-ene-3,l7-dione to the action of Pseudomonas s.p. B-20-l 84.

It would be, however, strongly desired in the art to find out the more advantageous method for the production of the estrane compound. As a result of our investigations on a method for the microbiological production of the estrane compound, it has been unexpectedly found that the l9-hydroxyandrostane compound (II) or (lll) can be satisfactorily converted to the estrane compound (I) by the action ofa microorganism selected from those micro-organisms of the genera C()) \'HI7t1(ffilflll and Arlhmhacter.

lt is, therefore, an object of this invention to provide a novel and advantageous method for the microbiological production of an estrane compound by the action of a micro-organism of the genera Corynebacterium or Arrhmbacler. Other objects of this invention will be apparent to those skilled in the art from the following disclosure.

in the microbiological method of this invention as depicted above, typical examples of the l9-hydroxyandrostane compound (II) and (lll) to be employed as a substrate include the following steroid:

l9-hydroxyandrost-4-ene-3, l 7-dionie,

3, l 9-dihydroxyandrost-5-enl 7-one and l7a-ethynyl- 1 7B, 1 9-dihydroxyandrost-4-en-3-one.

Typical examples of the micro-organisms which have been found to be useful for the method of this invention include those micro-organisms: that is,

Corynebacterium equi and Arthrobacter ureafaciens These micro-organisms are described in the Bergeys Manual of Determinative Bacteriology, 7th Edition, p.588 1957).

in carrying out the method of this invention, with such a micro-organisms as describedabove as such may be inoculated a suitable culture medium containing said compound (ll) or (III) as a substrate, and then cultivation may be ef fected under aerobic condition. The method may also be carried out by employing the above-mentioned micro-organisms after adaption. Thus, the culture in which said micro-organisms had previously incubated in a suitable medium containing said compound (ll) or (ill) may be incorporated and cultivated in a suitable culture medium containing said compound (II) or (ill) as a substrate under aerobic condition. Alternatively, the micro-organism may be inoculated and cultivated in a suitable culture medium containing no substrate under aerobic condition and successively the aerobic culture is conducted after addition of the substrate. Moreover, the method of this invention may be successfully conducted by employing the enzymes (or the crude liquor thereof) obtained from the growing micro-organism through disruption of the cell by a conventional means, for example, by a French Press, a sonic oscillator, lyzozyme, a surface active agent and the like. In any case, the aerobic condition should be kept in the present method. The culture medium may be composed of usual ingredients commonly used for the cultivation of such micro-organisms as described above. Suitable culture medium contains a source of carbon, nitrogen and, if necessary, mineral elements (inorganic salts). Suitable carbon sources include glucose, sucrose, xylose, cane sugar, starch, glycerin and the like. Suitable nitrogen sources include corn steep liquor, peptone, yeast extract, meat extract, soybean meal and the like. Suitable mineral elements sulfate, calcium sodium chloride, ammonium nitrate, magnesium sulfate, calcium carbonate, dipotassium hydrogenphosphate and the like.

in carrying out the fermentation, there may be satisfactorily employed any of aerobic culture procedures commonly used in the art, but shaking culture, stationary culture and culture with aeration may be preferably employed. The culture is generally conducted at a temperature of about 25 C. to 30 C. It is desirable that the pH value of the culture medium is within the pH range of about 5.0 to 9.0, preferably about 6.0 to 8.0. The fermentation is generally continued for about 3 to 15 days. The substrate may be added to the culture medium either in a finely divided form or as a solution (suitably, a saturated solution) in a suitable organic solvent, such as dimethylformamide, methanol, acetone and the like. It has also been found that, when Corynebacterium equi is employed as a micro-organism, addition of the substrate to a culture medium can be effected in a high amount, preferably about 2-3 w/v percent, thereby to improve the yield of the desired estrane compound. in this case, it is desirable that a surface active agent of fatty acid ester series, such as Tween 20, 60, and Span 60; and Myrj 49 and 51 (these are trade names of the surface active agents manufactured and sold by Atlas Chemical Industries Wilmington 99, Del. U.S.A.) is added to the culture medium at a concentration of about 3 w/v percent, so that the substrate is well dispersed in a culture medium and an activity of the surface of the substrate particle is also elevated. During the cultivation, a carbon source may be additionally supplied by optional addition of sugar such as glucose and the like to shorten the cultivation period and to suppress the production of byproducts. When sugar, e.g., glucose is added, the pH of a culture medium is inclined to acidic. Thus, it is preferable to maintain the pH of a culture medium over pH 5.0 by addition ofa proper amount of calcium carbonate.

in the process of this invention, where there is employed as a substrate the l9-hydroxyandrostane compound of the formula (II) or (III) wherein the R is hydroxy group and the R is hydrogen atom, there is obtained the estrane compound of the formula (I) wherein the R and the R together with the carbon atom to which they are attached, represent keto group. The isolation of the desired product from the fermentation broth may be conducted by any suitable manner well known in the art. For instance, the broth is extracted several times with ethyl acetate, the combined extracts are washed successively with aqueous sodium bicarbonate, aqueous dilute hydrochloric acid and water, dried over anhydrous sodium sulfate and concentrated to obtain crude crystalline substance. The crude crystalline substance is recrystallized from a suitable solvent such as acetone, methylene chloride or chromatographed through a column of alumina with ethylacetate-n-hexane to give the desired compound (I).

The following examples are given solely for the purpose of illustration of this invention, but they should not be construed as limiting the scope thereof.

EXAMPLE I A culture medium comprising the following ingredients was prepared:

The culture medium (200 ml.) was adjusted to pH 7.2-7.4 and divided into two SOO-mL-volume shaking flasks, each containing 100 ml. of the medium. After sterilization at 120 C. (under pressure of lb.) for 15 minutes, a substrate, i.e., I9- hydroxy-androst-4-ene-3,l7-dione dissolved in 2 ml. of dimethylformamide at the concentration indicated hereinbelow was added with each 1 ml. portion to these flasks. Then Corynebacrerium equi was inoculated and shaking culture was carried out at 30 C. with 120 r.p.m. for from 3 to II days. After incubation, the fermentation broths were collected and extracted three times with ethyl acetate. The combined extracts were concentrated under reduced pressure to give oily substance. The oily substance was then chromatographed through a column of alumina by employing as an eluent ethyl acetate-cyclohexane. After removal of the solvent from the eluate, there was obtained the final product, estrone.

The product thus obtained was identified with an authentic specimen by means of a position on thin layer chromatography, chemical color tests and ultraviolet absorption spectra.

By employing Arrhrobacter ureafaciens instead of Corynebacterium equi, there was similarly obtained estrone. The yields of estrone obtained by the above-mentioned method were as follows:

EXAMPLE 2 TABLE 2 Percent Concentration of the substrate added. w./v. percent 0.05 0. l

Microorganism:

Corunebacterium equi 23. 3 25.0 Arthrobacter ureafaciem 73. 5 76. 0

EXAMPLE 3 To a 50-ml.-volume shaking flask having the shape of the letter L" was placed 20 ml. of a main culture medium (pH 6.2) comprising peptone 2 percent, corn steep liquor 0.5 percent and Tween 3.0 percent which is sterilized at 120 C. for 15 minutes and then allowed to cool. To the cooled medium was incorporated 400 mg. (2 g. per ml. of the medium) of a finely divided substrate, l9-hydroxyandrost-4-ene- 3,17-dione (I), having been previously sterilized by irradiation of ultraviolet ray.

On the other hand, a liquid culture medium (pH 7.2) comprising peptone 2 percent and corn steep liquor 0.5 percent and inoculated with Corynebacterium equi was subjected to a shaking culture at 26 C. for 24 hours.

With 2 ml. of the seed culture broth obtained as described above was inoculated the above-mentioned main culture medium containing the substrate.

Then, a shaking culture was conducted at 26 C. for 24 hours. At the end of this time, 100 mg. of calcium carbonate and 0.5 ml. of a 40 percent aqueous glucose solution were aseptically incorporated into the main medium (final concentration of glucose in the medium was 1.0 percent).

A shaking culture was continued for additional 24 hours. At the end of this time 0.5 ml. of a 40 percent aqueous glucose solution was again incorporated into the medium (final concentration ofglucose in the medium was 1.0 percent).

Then, a shaking culture was effected for additional 24 hours. At the end of this time, the substrate powder incorporated disappeared away and, instead, estrone were separated as needles in the fermentation broth. Such oxidative fermentation has come to an end in 3 days.

The fermentation broth was extracted several times with ethyl acetate, the combined extracts were concentrated under reduced pressure, and the residue was chromatographed through a column of alumina to yield estrone as crystals in a yield of about 78 percent.

The product so obtained was identified with an authentic specimen by means ofa thin layer chromatography. It melts at 250-252 C., and shows )tmax EtoH 28] my and [a]D20+l59.

EXAMPLE 4 Following the procedure of example 3, 500 mg. (2.5 g. per 100 ml. of the main culture medium) of 3,l9-dihydroxyandrost-5-en-l7-one (II) was incorporated into the main culture medium and then the seed culture broth containing Corynebacterium equi was added thereto.

Then, a shaking culture was conducted at 26 C. for 24 hours. At the end of this time, 100 mg. of calcium carbonate and 0.5 ml. of a 40 percent aqueous glucose solution were aseptically incorporated into the fermentation broth (final concentration of glucose in the medium was l.0 percent).

Thereafter. a shaking culture was continued in the same manner as above. for 4 days, during which period of time was incorporated each 0.5 ml. portion of a 40 percent aqueous glucose solution every 24 hours. The oxidative fermentation has come to an end in 5 days. The fermentation broth was extracted several times with ethyl acetate, the combined extracts were concentrated under reduced pressure and the residue was chromatographed through a column of alumina as described in example 4, thereby to yield estrone as crystals in a yield of about 69 percent. The product so obtained melts at 24725l C. and shows Amax EtoH 281 my. and [a]D=+ l63.

EXAMPLE 5 The same procedure as in example 3 is repeated except the calcium carbonate and aqueous glucose solution were not incorporated into the main culture medium during the fermentation. There was obtained estrone as crystals in a yield of 57 percent.

EXAMPLE 6 TABLE 3 Percent Concentration of the substrate added, w./v. percent 0. 1 0. 05

Microorganism:

Corynebacterium equi 28. 4

Arthrobacter meafaciens 33. 7

What is claimed is: l. A method for the production of a compound having the formula wherein R is a hydroxyl group and R is a lower alkynyl group or R and R when taken together with the carbon atom to which they are attached, represent a keto group which comprises subjecting a starting compound having the formula wherein R is a hydroxyl group and R is hydrogen or a lower alkynyl group or R and R when taken together with the carbon atom to which they are attached. represent a keto group to the action of Corynebacterium equu' or to the action of the enzyme produced by said micro-organism under aerobic conditions in the presence of a surface active agent of the fatty acid ester series, said starting compound being incorporated into a culture medium at a concentration of 2 to 3 w/v percent.

2. The method according to claim 1 wherein said surface active agent is Tween 80.

3. The method according to claim 1 wherein said starting compound is selected from the compounds consisting of 19- hydroxyandrost-4-ene-3,l7-dione, 3.,l9-dihydroxyandrost-5- en-17-one, and l7a-ethynyl-ll7B,l9-dihydroxyandrost-4-en-3- one. 

2. The method according to claim 1 wherein said surface active agent is Tween
 80. 3. The method according to claim 1 wherein said starting compound is selected from the compounds consisting of 19-hydroxyandrost-4-ene-3,17-dione, 3,19-dihydroxyandrost-5-en-17-one, and 17 Alpha -ethynyl-17 Beta ,19-dihydroxyandrost-4-en-3-one. 